CN117571882B - Liquid chromatography-tandem mass spectrometry detection method for steroid hormone in serum - Google Patents
Liquid chromatography-tandem mass spectrometry detection method for steroid hormone in serum Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 27
- 239000003270 steroid hormone Substances 0.000 title claims abstract description 24
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 title claims abstract description 17
- 210000002966 serum Anatomy 0.000 title claims abstract description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 114
- 239000003480 eluent Substances 0.000 claims abstract description 53
- 238000000034 method Methods 0.000 claims abstract description 32
- 239000000243 solution Substances 0.000 claims abstract description 29
- 239000007864 aqueous solution Substances 0.000 claims abstract description 26
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
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- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims abstract description 10
- 235000011114 ammonium hydroxide Nutrition 0.000 claims abstract description 10
- 235000019253 formic acid Nutrition 0.000 claims abstract description 10
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- AUALQMFGWLZREY-UHFFFAOYSA-N acetonitrile;methanol Chemical compound OC.CC#N AUALQMFGWLZREY-UHFFFAOYSA-N 0.000 claims abstract description 4
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 claims description 32
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- CZWCKYRVOZZJNM-USOAJAOKSA-N dehydroepiandrosterone sulfate Chemical compound C1[C@@H](OS(O)(=O)=O)CC[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CC=C21 CZWCKYRVOZZJNM-USOAJAOKSA-N 0.000 claims description 17
- 229950009829 prasterone sulfate Drugs 0.000 claims description 17
- 150000001875 compounds Chemical class 0.000 claims description 16
- 229960000890 hydrocortisone Drugs 0.000 claims description 16
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- NVKAWKQGWWIWPM-ABEVXSGRSA-N 17-β-hydroxy-5-α-Androstan-3-one Chemical compound C1C(=O)CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CC[C@H]21 NVKAWKQGWWIWPM-ABEVXSGRSA-N 0.000 claims description 13
- DDFHBQSCUXNBSA-UHFFFAOYSA-N 5-(5-carboxythiophen-2-yl)thiophene-2-carboxylic acid Chemical compound S1C(C(=O)O)=CC=C1C1=CC=C(C(O)=O)S1 DDFHBQSCUXNBSA-UHFFFAOYSA-N 0.000 claims description 13
- MUMGGOZAMZWBJJ-DYKIIFRCSA-N Testostosterone Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 MUMGGOZAMZWBJJ-DYKIIFRCSA-N 0.000 claims description 12
- FMGSKLZLMKYGDP-USOAJAOKSA-N dehydroepiandrosterone Chemical compound C1[C@@H](O)CC[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CC=C21 FMGSKLZLMKYGDP-USOAJAOKSA-N 0.000 claims description 12
- 229960003473 androstanolone Drugs 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- FMGSKLZLMKYGDP-UHFFFAOYSA-N Dehydroepiandrosterone Natural products C1C(O)CCC2(C)C3CCC(C)(C(CC4)=O)C4C3CC=C21 FMGSKLZLMKYGDP-UHFFFAOYSA-N 0.000 claims description 9
- 238000002386 leaching Methods 0.000 claims description 9
- 229960002847 prasterone Drugs 0.000 claims description 9
- DBPWSSGDRRHUNT-UHFFFAOYSA-N 17alpha-hydroxy progesterone Natural products C1CC2=CC(=O)CCC2(C)C2C1C1CCC(C(=O)C)(O)C1(C)CC2 DBPWSSGDRRHUNT-UHFFFAOYSA-N 0.000 claims description 6
- AEMFNILZOJDQLW-QAGGRKNESA-N androst-4-ene-3,17-dione Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CCC2=C1 AEMFNILZOJDQLW-QAGGRKNESA-N 0.000 claims description 6
- 229960005471 androstenedione Drugs 0.000 claims description 6
- AEMFNILZOJDQLW-UHFFFAOYSA-N androstenedione Natural products O=C1CCC2(C)C3CCC(C)(C(CC4)=O)C4C3CCC2=C1 AEMFNILZOJDQLW-UHFFFAOYSA-N 0.000 claims description 6
- 229960003604 testosterone Drugs 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 5
- 238000004811 liquid chromatography Methods 0.000 claims description 5
- 229950000801 hydroxyprogesterone caproate Drugs 0.000 claims description 4
- DOMWKUIIPQCAJU-LJHIYBGHSA-N Hydroxyprogesterone caproate Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@@](C(C)=O)(OC(=O)CCCCC)[C@@]1(C)CC2 DOMWKUIIPQCAJU-LJHIYBGHSA-N 0.000 claims 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 6
- 150000003904 phospholipids Chemical class 0.000 abstract description 6
- 230000035945 sensitivity Effects 0.000 abstract description 5
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- 239000000047 product Substances 0.000 description 22
- 238000003908 quality control method Methods 0.000 description 21
- 238000002360 preparation method Methods 0.000 description 16
- DBPWSSGDRRHUNT-CEGNMAFCSA-N 17α-hydroxyprogesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@@](C(=O)C)(O)[C@@]1(C)CC2 DBPWSSGDRRHUNT-CEGNMAFCSA-N 0.000 description 15
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- UOXJNGFFPMOZDM-UHFFFAOYSA-N 2-[di(propan-2-yl)amino]ethylsulfanyl-methylphosphinic acid Chemical compound CC(C)N(C(C)C)CCSP(C)(O)=O UOXJNGFFPMOZDM-UHFFFAOYSA-N 0.000 description 1
- -1 COR Chemical class 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
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- Life Sciences & Earth Sciences (AREA)
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- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention relates to the technical field of analysis and detection, in particular to a liquid chromatography-tandem mass spectrometry detection method for steroid hormone in serum. The method comprises the following steps: mixing a sample to be detected with an internal standard substance and a methanol aqueous solution, and separating to obtain a supernatant; after purifying the supernatant by adopting the PEP plate, eluting the PEP plate by adopting eluent A and eluent B in sequence; the eluent A is methanol aqueous solution containing ammonia water; the eluent B is methanol aqueous solution containing formic acid; eluting the PEP plate by using eluent C, and collecting eluent; eluent C is acetonitrile methanol solution; mixing the eluent with water, and detecting by adopting liquid chromatography-tandem mass spectrometry. The pretreatment process of the method can greatly reduce the content of phospholipid in the sample to be tested, and remarkably improve the testing accuracy and sensitivity of the sample; the pretreated sample can be detected on the machine without nitrogen blowing and re-dissolving, and the detection efficiency is greatly improved.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a liquid chromatography-tandem mass spectrometry detection method for steroid hormone in serum.
Background
Hormone (Hormone) is also called Hormone, and refers to a chemical substance generated by a certain cell, gland or organ in the body and capable of affecting the activities of other cells in the body, and the metabolism of the cells can be changed by only a small dosage of Hormone. Steroid hormones, also known as steroid hormones, are secreted by the gonads and adrenal glands, including androgens, estrogens and adrenal corticoids, are secreted in extremely small amounts and vary with age and sex, generally on the order of nmol/L and pmol/L, but are extremely important in their action and have a wide regulatory role in maintaining life, growth and development, fertility, inflammation, tumorigenesis, and mood control. The endocrine system of the human body secretes various hormones, and together with the nervous system, regulates the metabolism and physiological functions of the human body. Under normal conditions, various hormones are in balance, and if endocrine disturbance is caused by the balance being broken for some reason, the endocrine disturbance can have adverse effects on the body and spirit, and even endanger life, so that the steroid hormone level is clinically measured as a diagnostic reference index of many diseases.
For ultra-micro, high-sensitivity, accurate quantitative detection of steroid hormones in vivo, liquid chromatography-tandem mass Spectrometry (Liquid Chromatography TANDEM MASS Spectrometry, LC-MS/MS) has been recently internationally preferred. The phospholipid content in the sample to be detected can significantly influence the accuracy and sensitivity of liquid chromatography-tandem mass spectrometry detection.
In the prior art, CN 111398446A provides a method for detecting 12 steroid hormones in serum by using an ultra-high performance liquid chromatography-tandem mass spectrometry technology, but the pretreatment adopts a liquid phase extraction mode, the impurity removal is not clean enough, the extracted supernatant has a large volume, the sample can be dried only by taking longer nitrogen blowing time, and the pretreatment time is longer, so that the method is not suitable for clinically rapidly detecting a large number of samples. CN 112051348A discloses a solid phase extraction method of steroid hormones in serum or plasma, and the pretreatment method still has poor effect on removing phospholipids.
Disclosure of Invention
In order to solve the defects in the prior art, after a large amount of fumbling attempts are carried out on a combination of a solid phase extraction plate, a leaching solution and an eluent, the PEP plate is selected as a solid phase extraction matrix, a methanol aqueous solution containing ammonia water with a specific concentration and a methanol aqueous solution containing formic acid with a specific concentration are adopted as the leaching solution in sequence, and acetonitrile methanol solution with a specific proportion is further adopted as the eluent for purifying a sample, so that the content of phospholipid in the sample to be detected entering the liquid chromatograph can be greatly reduced, and meanwhile, the enrichment effect of 7 steroid hormones such as Cortisol (COR), testosterone (T), 17-alpha-hydroxyprogesterone (17-OHP), dihydrotestosterone (DHT), androstenedione (A4), dehydroepiandrosterone (DHEA) and dehydroepiandrosterone-sulfate (DHEAS) is good, so that the test accuracy and the sensitivity of the sample are remarkably improved.
Based on this, the following summary is specifically proposed.
First, the present invention provides a method for detecting steroid hormone, comprising:
(1) Mixing a sample to be detected with an internal standard substance and a methanol aqueous solution, and separating to obtain a supernatant;
(2) Purifying the supernatant by adopting PEP plates, and sequentially leaching the PEP plates by adopting leacheate A and leacheate B;
the leaching solution A is a methanol aqueous solution containing ammonia water; wherein, the ammonia water accounts for 0.1 to 0.15 percent of the volume of the eluent A, and the methanol accounts for 30 to 40 percent of the volume of the eluent A;
the leaching solution B is methanol aqueous solution containing formic acid; wherein, the formic acid accounts for 0.1 to 0.15 percent of the volume of the eluent B, and the methanol accounts for 30 to 40 percent of the volume of the eluent B;
(3) Eluting the PEP plate by using eluent C, and collecting eluent; the eluent C is acetonitrile methanol solution; wherein acetonitrile accounts for 80-85% of the volume of the eluent C;
(4) Mixing the eluent with water, and detecting by adopting liquid chromatography-tandem mass spectrometry.
Preferably, in the eluent A, the ammonia water accounts for 0.1-0.12% of the volume of the eluent A, and the methanol accounts for 32-38% of the volume of the eluent A.
Preferably, in the eluent B, formic acid accounts for 0.1-0.12% of the volume of the eluent B, and methanol accounts for 32-38% of the volume of the eluent B.
The PEP plate is leached by adopting the two leaches with the content, so that the removal effect of the phospholipid is better.
Preferably, in the eluent C, acetonitrile accounts for 82% -85% of the volume of the eluent C.
The eluting solution C with the content has better enriching effect on the 7 steroid hormone.
Preferably, the PEP sheet is activated with methanol and water in sequence prior to use.
Preferably, in step (4), the volume ratio of the eluent to water is 1: (0.8-1.5) and mixing.
Preferably, the detection conditions of the liquid chromatography include:
The chromatographic column is a C18 column, the specification is (2-3) × (80-100) mm, and the specification is (3-5) μm;
mobile phase a is an aqueous solution containing ammonium fluoride and mobile phase B is a methanol solution containing ammonium fluoride.
Preferably, the elution procedure of the liquid chromatography is:
in the elution process, the sum of the volume percentages of the mobile phase A and the mobile phase B is 100%.
The invention further aims at the sample after pretreatment, optimizes the mobile phase and the elution program in a matching way, and discovers that under the combination condition of the liquid chromatograph, the peak type and the response value of the steroid hormone can be better.
Preferably, mobile phase a comprises a volume ratio of 1L: (0.3-0.7) mL of water and ammonium fluoride aqueous solution; the concentration of the ammonium fluoride aqueous solution is 0.03-0.08 mM.
More preferably, mobile phase a comprises a volume ratio of 1L: (0.4-0.6) mL of water and ammonium fluoride aqueous solution; the concentration of the ammonium fluoride aqueous solution is 0.04-0.06 mM.
Preferably, mobile phase B comprises a volume ratio of 1L: (0.3-0.7) mL of an aqueous solution of methanol and ammonium fluoride; the concentration of the ammonium fluoride aqueous solution is 0.03-0.08 mM.
More preferably, mobile phase B comprises a volume ratio of 1L: (0.4-0.6) mL of an aqueous solution of methanol and ammonium fluoride; the concentration of the ammonium fluoride aqueous solution is 0.04-0.06 mM.
Preferably, the flow rate in the elution process is 0.4-0.8 mL/min; and/or the column temperature of the chromatographic column is 38-42 ℃.
Preferably, the steroid hormone is at least one of Cortisol (COR), testosterone (T), 17-alpha-hydroxyprogesterone (17-OHP), dihydrotestosterone (DHT), androstenedione (A4), dehydroepiandrosterone (DHEA), dehydroepiandrosterone Sulfate (DHEAs).
Preferably, the internal standard is a deuterated compound of the steroid hormone.
The compound isotope is used as an internal standard substance, so that endogenous interference can be eliminated, meanwhile, the compound isotope is similar to the compound physical substance, has certain stability under the same pretreatment and chromatographic conditions, and errors caused by changes of operation conditions and the like are eliminated to a certain extent.
Preferably, the liquid chromatography-tandem mass spectrometry is a high performance liquid chromatography-tandem mass spectrometry.
In some embodiments, the detection method further comprises:
and replacing the sample to be detected with a quality control product or a calibrator for detection.
In some embodiments, the sample to be tested is human serum or plasma.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a liquid chromatography-tandem mass spectrometry detection method of steroid hormone in serum, wherein the pretreatment process of the method can greatly reduce the content of phospholipid in a sample to be detected, and the test accuracy and the sensitivity of the sample are obviously improved; the pretreated sample can be detected on the machine without nitrogen blowing and re-dissolving, and the detection efficiency is greatly improved. The detection method can meet the requirements of various aspects such as linear range, repeatability, batch-to-batch difference, accuracy and the like.
Drawings
FIG. 1 is a chromatogram of cortisol (left panel) and its internal standard (right panel).
Fig. 2 is a chromatogram of testosterone (left panel) and its internal standard (right panel).
Figure 3 is a chromatogram of 17-hydroxyprogesterone (left panel) and its internal standard (right panel).
Fig. 4 is a chromatogram of dihydrotestosterone (left panel) and its internal standard (right panel).
FIG. 5 is a chromatogram of androstenedione (left panel) and its internal standard (right panel).
FIG. 6 is a chromatogram of dehydroepiandrosterone (left panel) and its internal standard (right panel).
FIG. 7 is a chromatogram of dehydroepiandrosterone sulfate (left panel) and its internal standard (right panel).
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The examples are not intended to identify the particular technology or conditions, and are either conventional or are carried out according to the technology or conditions described in the literature in this field or are carried out according to the product specifications. The reagents and instruments used, etc. are not identified to the manufacturer and are conventional products available for purchase by regular vendors.
Brand of PEP plate: ai Jieer; cargo number: PE00501-MW.
The instrument model of the liquid chromatograph-tandem mass spectrometer is as follows: AB SCIEX Triple QuadTM 4500MD.
In the following examples, 0.5% bovine serum albumin was selected as a substitute matrix for human serum samples to prepare various concentration points and quality control of standard curves, and the standard curves have good stability, are easy to obtain and convenient to store.
Example 1
The embodiment provides a high performance liquid chromatography-tandem mass spectrometry detection method for simultaneously detecting 7 steroid hormones of Cortisol (COR), testosterone (T), 17-alpha-hydroxyprogesterone (17-OHP), dihydrotestosterone (DHT), androstenedione (A4), dehydroepiandrosterone (DHEA) and dehydroepiandrosterone-sulfate (DHEAS) in human serum, which comprises the following specific steps:
Preparation of Compound stock solution and Secondary stock solution
1. The purchased T, DHEAS, 17-OHP, DHT, A, COR, DHEA standards were all 1mg/mL, each of which was precisely removed and methanol was used as a diluent to prepare a secondary stock solution 1 according to the following table 1, wherein DHEAs was not diluted and stored in a refrigerator at-20 ℃ for use, for example, 1 mL.
Formulation of Compound Secondary stock solution 1
2. The secondary stock solution 1 prepared in Table 1 was precisely removed, and methanol was used as a diluent to prepare a mixed secondary stock solution 2, for use, as shown in Table 2 below, using 1mL as an example.
Formulation of compound mix secondary stock solution 2 of table 2
(II) preparation of internal standard stock solution and internal standard extract
1. The purchased T-d3, DHEAS-d6, 17-OHP-d8, DHT-d4, A4-d7, COR-d4 and DHEA-d6 standard substances are precisely weighed respectively, methanol is used as a diluent, a volumetric flask is used for constant volume, the purity of the standard substances, the carried crystal water or salt (if any) is converted during actual weighing, and stock solutions with the concentration of 1000 mug/mL of T-d3, DHEAS-d6, 17-OHP-d8, DHT-d4, A4-d7, COR-d4 and DHEA-d6 are obtained respectively and are stored in a refrigerator at the temperature of minus 20 ℃ for standby.
2. The stock solutions were precisely removed, methanol was used as a diluent, and an internal standard secondary stock solution was prepared as shown in Table 3 below, and stored in a-20℃refrigerator for use, for example, 1 mL.
TABLE 3 preparation of internal Standard secondary stock solutions
3. The secondary stock solution 1 of the internal standard prepared in Table 3 was precisely removed, and methanol was used as a diluent to prepare an internal standard extract according to the following Table 4, taking 100mL as an example.
Table 4 preparation of internal standard extracts
(III) preparation of calibration materials
1. Preparation of 0.5% BSA solution
Taking 100mL as an example:
0.5g of bovine serum albumin was weighed to 100mL of 1 XPBS solution with a balance and thoroughly mixed for further use.
2. Preparation of calibration Material 6
Taking 100mL of calibrator 6 as an example:
the mixed secondary stock solution 2 in Table 2 was precisely removed by taking 5mL, and then added with 0.5% BSA solution to a volume of 100mL, and the mixture was turned upside down at least 10 times and mixed uniformly as shown in Table 5 below.
Table 5 preparation of calibrator 6
3. Blank liquid H0 preparation
Taking 100mL of blank solution H0 as an example:
Precisely removing 5mL of methanol, adding 0.5% BSA solution to 100mL, reversing at least 10 times, and mixing uniformly.
4. Preparation of calibration materials 1-6
Taking 50.00mL of calibration materials 1-5 as an example, the calibration materials 1-5 are prepared by respectively diluting the calibration material 6 and the blank liquid H0, and the dilution ratio is shown in Table 6.
Table 6 preparation of calibration materials 1-5
(IV) preparation of quality control product
Taking 50.00mL of quality control products 1-3 as an example, the quality control products 1-3 are prepared by respectively diluting a calibrator 6 and a blank solution H0, wherein the dilution ratio is shown in Table 7.
Table 7 quality control product 1-3 preparation
(V) sample pretreatment method
1. Reagent preparation:
Eluent A: an aqueous solution containing 0.1% ammonia water and 35% methanol by volume;
eluent B: an aqueous solution containing 0.1% formic acid by volume and 35% methanol;
eluent C: the volume ratio is 85:15 in acetonitrile and methanol;
mobile phase additive: ammonium fluoride aqueous solution at a concentration of 0.05 mM.
2. Pretreatment:
(1) Pretreatment: all the calibrator, quality control and human serum samples are respectively taken into 400 mu L to 2mL centrifuge tubes, 400 mu L of internal standard extract is respectively added, the mixture is uniformly mixed for 10min at 2000rpm by vortex, then 400 mu L of water is added, the mixture is uniformly mixed for 5min at 2000rpm by vortex, finally the mixture is centrifuged for 5min at 4 ℃ and 12000rpm, and the supernatant is ready for use.
(2) PEP plate activation: the PEP plate was activated with 300. Mu.L of methanol and 300. Mu.L of water in this order, and the waste liquid was discarded.
(3) Loading a sample: 800. Mu.L of the supernatant after the pretreatment in step 1) was slowly passed through the activated PEP plate and the waste liquid was discarded.
(4) Leaching: the PEP plate was rinsed with 300. Mu.L of eluent A and 300. Mu.L of eluent B in sequence, and the waste liquid was discarded.
(5) Eluting: a new 96-well plate is placed below the PEP plate, 60 mu L of eluent C is used for eluting, 50 mu L of water is added after eluting, the mixture is uniformly mixed, and the mixture is centrifuged at 4000rpm for 5min, so that the detection can be carried out on the machine.
(6) And (3) detection: and (5) detecting on-machine by a liquid chromatograph-tandem mass spectrometer.
(Six) liquid chromatography-tandem Mass Spectrometry conditions
1. The liquid chromatography conditions are shown in Table 8.
TABLE 8 liquid chromatography conditions
2. The mass spectrum conditions are shown in tables 9 and 10.
TABLE 9 ion Source parameters
Table 10 ion pair parameters
The chromatograms of cortisol, testosterone, 17-hydroxyprogesterone, dihydrotestosterone, androstenedione, dehydroepiandrosterone sulfate and internal standards thereof are respectively shown in figures 1-7.
Example 2
This example demonstrates the methodology of example 1.
1. Linearity of
(1) The verification method comprises the following steps: 3 batches of standard curves were prepared continuously, and the relative deviation and linear correlation coefficient of each concentration point were calculated by detecting the standard curves by the detection method of example 1. The correlation coefficient r of the linear regression is calculated according to the following formula and data analysis software.
(2) Acceptance criteria: the deviations of the linear minimum concentration points of COR, T,17-OHP, DHT, A4, DHEA and DHEAS are all within +/-20.0%, the deviations of the other concentration points are all within +/-15.0%, and the correlation coefficient r of linear regression is more than or equal to 0.990.
(3) The experimental results are shown in tables 11 to 17.
TABLE 11 Linear results of Compound COR
TABLE 12 Linear results for Compound T
TABLE 13 Linear results of Compound 17-OHP
TABLE 14 Linear results of Compound DHT
TABLE 15 Linear results for Compound A4
TABLE 16 Linear results of the Compound DHEA
TABLE 17 Linear results of Compound DHEAS
(4) Conclusion: the deviations of the linear minimum concentration points of COR, T,17-OHP, DHT, A4, DHEA and DHEAS are within +/-20.0%, the deviations of the other concentration points are within +/-15.0%, and the correlation coefficient r of linear regression is more than or equal to 0.990, so that the acceptance standard is met.
2. Accuracy of method (recovery rate of adding mark)
(1) The verification method comprises the following steps: the method comprises the steps of adding a high-level object to be detected A solution with known concentration (7 compounds including COR, T,17-OHP, DHT, A4, DHEA and DHEAS), wherein the solution A can directly select a proper high-concentration reference substance, and if no reference substance exists, the solution A can also be prepared by a pure product consistent with a measured object, a weight method is adopted during the preparation to reduce uncertainty in the preparation process), adding the solution A solution into a serum sample B solution of a human, and preparing at least 3 recovered samples with different concentrations, wherein the volume ratio between the added object to be detected A solution and the serum sample B solution of the human is 1:9, calculating the recovery rate, wherein the recovery rate (R) of each compound of each sample is in the range of 85% -115%.
R=（C Actual measurement value -C Background value ）/ C Theoretical value ×100%
R: recovery rate;
C Actual measurement value : average of the measured values calculated from the linear fitting;
C Background value : background value of human serum sample;
c Theoretical value : theoretical concentration of the sample after formulation.
(2) Acceptance criteria: recovery (R) of COR, T,17-OHP, DHT, A4, DHEA and DHEAS should be in the range of 85% -115%.
(3) The experimental results are shown in table 18.
Table 18 results of the recovery rate by adding the standard
(4) Conclusion: the accuracy is high, and the recovery rate of low-concentration samples and high-concentration samples of COR, T,17-OHP, DHT, A4, DHEA and DHEAS are all within 85-115%.
3. Repeatability of
(1) The verification method comprises the following steps: under the condition of repeatability, the test is repeated 10 times for the quality control products 1-3 according to the detection method of the embodiment 1. The Coefficient of Variation (CV) of the reproducibility was calculated according to the following formula.
；
CV: a coefficient of variation of repeatability;
: average of 10 measurements;
S: standard deviation of 10 measurements.
(2) Acceptance criteria: the coefficient of variation CV of the quality control product 1 is less than or equal to 20 percent, and the coefficient of variation CV of the quality control products 2 and 3 is less than or equal to 15 percent.
(3) The experimental results are shown in table 19.
TABLE 19 repeatability results
(4) Conclusion: the coefficient of variation CV of the quality control product 1 is less than or equal to 20 percent, the coefficient of variation CV of the quality control products 2 and 3 is less than or equal to 15 percent, and the acceptance standard is met.
4. Difference between batches
(1) The verification method comprises the following steps: quality control products 1-3 prepared in different batches were tested 10 times respectively for 30 times according to the detection method of example 1. The Coefficient of Variation (CV) of the reproducibility was calculated according to the following formula.
；/>
CV: a coefficient of variation of repeatability;
: average of 30 measurements;
S: standard deviation of 30 measurements.
(2) Acceptance criteria: the coefficient of variation CV of the quality control product 1 is less than or equal to 20 percent, and the coefficient of variation CV of the quality control products 2 and 3 is less than or equal to 15 percent.
(3) The experimental results are shown in table 20.
TABLE 20 results of differences between batches
(4) Conclusion: the coefficient of variation CV of the low-value quality control product is less than or equal to 20 percent, and the coefficient of variation CV of the high-value quality control product is less than or equal to 15 percent, thereby meeting the acceptance standard.
In summary, the detection method of embodiment 1 has good specificity, strong specificity, high analysis sensitivity and high accuracy. The deviation of the linear lowest concentration point is within +/-20.0%, the deviation of the other concentration points is within +/-15.0%, and the linear regression fit constant r is greater than 0.990; the recovery rate of the serum standard adding samples of the low-concentration and high-concentration people is within 85-115 percent; the Coefficient of Variation (CV) of the repeatability of the quality control product 1 is less than or equal to 20 percent, and the Coefficient of Variation (CV) of the repeatability of the quality control product 2-3 is less than or equal to 15 percent; the inter-batch Coefficient of Variation (CV) of the quality control product 1 is less than or equal to 20 percent, and the inter-batch Coefficient of Variation (CV) of the quality control products 2-3 is less than or equal to 15 percent, and all meet the acceptance standard.
Comparative example 1
This comparative example provides a method for detecting steroid hormone, which differs from example 1 only in that:
eluent A: an aqueous solution containing 0.05% by volume of aqueous ammonia and 35% by volume of methanol.
Comparative example 2
This comparative example provides a method for detecting steroid hormone, which differs from example 1 only in that:
eluent B: an aqueous solution containing 0.05% by volume of formic acid and 35% by volume of methanol.
Comparative example 3
This comparative example provides a method for detecting steroid hormone, which differs from example 1 only in that:
eluent C was replaced with 50 volume ratio: 50 acetonitrile in methanol.
Effect example
The sample pretreated by the detection method of comparative examples 1 to 3 was taken and the labeled recovery was measured by referring to the labeled recovery measurement method of example 1.
The test results are shown in Table 21.
Table 21
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (9)
1. A method for detecting steroid hormone, comprising:
(1) Mixing a serum sample to be detected with an internal standard substance and a methanol aqueous solution, and separating to obtain a supernatant;
(2) Purifying the supernatant by adopting PEP plates, and sequentially leaching the PEP plates by adopting leacheate A and leacheate B;
the leaching solution A is a methanol aqueous solution containing ammonia water; wherein, the ammonia water accounts for 0.1 to 0.15 percent of the volume of the eluent A, and the methanol accounts for 30 to 40 percent of the volume of the eluent A;
the leaching solution B is methanol aqueous solution containing formic acid; wherein, the formic acid accounts for 0.1 to 0.15 percent of the volume of the eluent B, and the methanol accounts for 30 to 40 percent of the volume of the eluent B;
(3) Eluting the PEP plate by using eluent C, and collecting eluent; the eluent C is acetonitrile methanol solution; wherein acetonitrile accounts for 80-85% of the volume of the eluent C;
(4) Mixing the eluent with water, and detecting by adopting liquid chromatography-tandem mass spectrometry;
The steroid hormone is at least one of cortisol, testosterone, 17-alpha-hydroxyprogesterone, dihydrotestosterone, androstenedione, dehydroepiandrosterone and dehydroepiandrosterone sulfate.
2. The detection method according to claim 1, wherein in the eluent A, ammonia water accounts for 0.1% -0.12% of the volume of the eluent A, and methanol accounts for 32% -38% of the volume of the eluent A.
3. The detection method according to claim 2, wherein in the eluent B, formic acid accounts for 0.1% -0.12% of the volume of the eluent B, and methanol accounts for 32% -38% of the volume of the eluent B.
4. The method of claim 1, wherein the PEP sheet is activated with methanol and water sequentially prior to use.
5. The method according to claim 1, wherein in the step (4), the volume ratio of the eluent to water is 1: (0.8-1.5) and mixing.
6. The method according to claim 1, wherein the detection conditions of the liquid chromatography include:
The chromatographic column is a C18 column, the specification is (2-3) × (80-100) mm, and the specification is (3-5) μm;
mobile phase a is an aqueous solution containing ammonium fluoride and mobile phase B is a methanol solution containing ammonium fluoride.
7. The method according to claim 6, wherein the elution procedure of the liquid chromatograph is:
；
in the elution process, the sum of the volume percentages of the mobile phase A and the mobile phase B is 100%.
8. The detection method according to claim 7, wherein a flow rate in the eluting process is 0.4 to 0.8 mL/min; and/or the column temperature of the chromatographic column is 38-42 ℃.
9. The method of claim 1, wherein the internal standard is a deuterated compound of the steroid hormone.
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